The application of embryonic stem (ES) cell therapy for the treatment of human degenerative diseases should be performed in nonhuman primates prior to human trials. The macaque is an ideal animal model for neural transplantation studies because the developmental potential of the grafted cells can be timely and precisely examined postmortem. As a prelude to in vivo studies, the developmental potential and cell lineage differentiation of monkey ES cells must be established in vitro. To this end, we propose to culture and characterize monkey ES cells free of feeder cells before differentiating them into high-percentage cholinergic and dopaminergic neurons, and to determine the pluripotency of monkey ES cells by single cell culture and spontaneously differentiation into cells of all three embryonic germ layers in vitro, thereby addressing the objectives of this PA (PA-99-086/-01-076, NOVEL APPROACHES TO ENHANCE STEM CELL RESEARCH). Aim 1 will characterize two monkey ES cell lines with an XX and XY karyotype that are cultured under feeder cell-free conditions. Rhesus monkey ES cells will be plated on Matrigel- or laminin-coated dishes and cultured in a serum-free medium supplemented with fibroblast growth factors (FGFs), laminin, serum replacement, or conditioned medium from cell cultures to sustain the pluripotency and undifferentiated states. The growth rate, karyotype normalcy, and undifferentiated state will be determined. Aim 2 will directionally differentiate monkey ES cells into dopaminergic and cholinergic neurons, and to differentiate single monkey ES cell into all three embryonic germ layers in vitro. Feeder cell-free monkey ES cell derivatives or clonally derived ES cells will be subjected to paradigms involving growth factor removal with additions of conditioned medium, extracts of monkey tissues, or precursors of the dopaminergic and cholinergic synthesis pathway. The efficiency of cell type differentiation will be determined by counting dopaminergic and cholinergic cells that will be identified by specific protein or gene markers. This is a revised application that is focused on understanding the directional differentiation of monkey ES cells in vitro into dopaminergic and cholinergic neurons for future transplantation studies with monkey models of Parkinson's or Alzheimer's diseases.